1. Field of the Invention
The present invention relates to a novel set of markers, microarrays containing these markers, and an expression signature comprising 16 genes or a subset thereof and the use thereof in determining the regulation status of IL-6/STAT3 signaling pathway in a cell sample or subject, as well as compositions for the detection thereof. The regulation status of IL-6/STAT3 signaling pathway in a cell sample or subject may be assayed based on the level of expression of one or more of these genes. More specifically, the invention provides a set of genes which can be used as biomarkers and as gene signatures for evaluating IL-6/STAT3 pathway regulation or deregulation in a sample; diagnostic and/of classification of a sample, e.g., tumor, as having a deregulated IL-6/STAT3 signaling pathway; determining whether an agent modulates the IL-6/STAT3 signaling pathway in a sample; predicting the response of a subject to an agent that modulates the IL-6/STAT3 signaling pathway; assigning treatment to a subject; and evaluating the pharmacodynamic effects of therapies designed to target the IL-6/STAT3 pathway. The gene expression signature may be used with companion algorithms to provide a quantitative measure of IL-6/STAT3 pathway activity. Expression of the provided biomarkers is preferably determined by RT-PCR using SYBR green, and the expression data analyzed and compared to a control sample by use of the Random Forest method.
2. Description of Related Art
The STAT (Signal Transducer and Activator of Transcription) family consists of seven mammalian members. Originally, STAT proteins were identified as intracellular signaling mediators of cytokine signals. Every STAT family member responds to a defined set of cytokines. Interestingly, STAT3 is known to be activated by IL-6 (Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer. 2009 November; 9(11):798-809).
STAT proteins are latent cytoplasmic transcription factors that require phosphorylation for nuclear retention. Engagement of IL-6 to its specific receptor IL-6R (IL-6 receptor) activates receptor-associated tyrosine kinase, such as Janus Kinase 2 (JAK2). Activated JAK2 in turn phosphorylates tyrosine residues in the cytoplasmic tail of the IL-6 receptor that function as docking sites for STAT3. JAK2 dependent phosphorylation of STAT3 leads to its homodimerization and nuclear translocation, where activated STAT3 function as transcriptional activator, inducing expression of target genes (Levy D E, Darnell J E Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002 September; 3(9):651-62).
IL-6/STAT3 has been implicated as crucial mediator for inflammatory response (Grivennikov S I, Karin M. Dangerous liaisons: STAT3 and NF-kappaB collaboration and crosstalk in cancer. Cytokine Growth Factor Rev. 2010 February; 21(1):11-9. Epub 2009 Dec. 16). Moreover, deregulated IL-6/STAT3 signaling has been associated with biological events such as embryonic development, programmed cell death, organogenesis, innate immunity, adaptive immunity and cell growth regulation in many organisms (Mankan A K, Greten F R. Inhibiting signal transducer and activator of transcription 3: rationality and rationale design of inhibitors. Expert Opin Investig Drugs. 2011 September; 20(9):1263-75. Epub 2011 Jul. 14). In addition, STAT3 plays an essential role in cancer initiation and progression by selectively inducing and maintaining a pro-carcinogenic inflammatory microenvironment (Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat Rev Cancer. 2009 November; 9(11):798-809).
Perturbation of the IL-6/STAT3 signaling pathway causes a change in STAT3 transcriptional activity and, in turn, alters the expression level of STAT3 target genes. Although changes in gene expression of STAT3 target genes can serve as indicators of IL-6/STAT3 pathway activity, real time PCR assay based methods are not yet available to quantitatively measure IL-6/STAT3 pathway activity.
The identification of patient subpopulations most likely to respond to therapy is a central goal of modem molecular medicine. This notion is particularly important for cancer due to the large number of approved and experimental therapies (Rothenberg et al., 2003, Nat. Rev. Cancer 3:303-309), low response rates to many current treatments, and clinical importance of using the optimal therapy in the first treatment cycle (Dracopoli, 2005, Curr. Mol. Med. 5:103-110). In addition, the narrow therapeutic index and severe toxicity profiles associated with currently marketed cytotoxics results in a pressing need for accurate response prediction. Although recent studies have identified gene expression signatures associated with response to cytotoxic chemotherapies (Folgueria et al., 2005, Clin. Cancer Res. 11:7434-7443; Ayers et al., 2004, 22:2284-2293; Chang et al., 2003, Lancet 362:362-369; Rouzier et al., 2005, Proc. Natl. Acad. Sci. USA 102: 8315-8320), these examples (and others from the literature) remain unvalidated and have not yet had a major effect on clinical practice. In addition to technical issues, such as lack of a standard technology platform and difficulties surrounding the collection of clinical samples, the myriad of cellular processes affected by cytotoxic chemotherapies may hinder the identification of practical and robust gene expression predictors of response to these agents. One exception may be the recent finding by microarray that low mRNA expression of the microtubule-associate protein Tau is predictive of improved response to paclitaxel (Rouzier et al., supra).
To improve on the limitations of cytotoxic chemotherapies, current approaches to dnig design in oncology are aimed at modulating specific cell signaling pathways important for tumor growth and survival (Hahn and Weinberg, 2002, Nat. Rev. Cancer 2:331-341; Hanahan and Weinberg, 2000, Cell 100:57-70; Trosko et al., 2004, Ann. N.Y. Acad. Sci. 1028:192-201). In cancer cells, these pathways become deregulated resulting in aberrant signaling, inhibition of apoptosis, increased metastasis, and increased cell proliferation (reviewed in Adjei and Hildalgo, 2005, J. Clin. Oncol. 23:5386-5403). Although normal cells integrate multiple signaling pathways for controlled growth and proliferation, tumors seem to be heavily reliant on activation of one or two pathways (“oncogene activation”). The components of these aberrant signaling pathways represent attractive selective targets for new anticancer therapies. In addition, responder identification for target therapies may be more achievable than for cytotoxics, as it seems logical that patients with tumors that are “driven” by a particular pathway will respond to therapeutics targeting components of that pathway. Therefore, it is crucial that methods to identify the pathways that are active in particular tumors are developed, and this information used to guide therapeutic decisions. Identification of gene expression profiles that are indicative of pathway activation status is one way to achieve this goal.
Given its involvement in numerous biological functions and diseases, a gene expression signature-based readout of IL-6/STAT3 pathway activation may be more appropriate than relying on a single indicator of pathway activity, as the same signature of gene expression may be elicited by activation of multiple components of the pathway.
Based on the foregoing, a reliable method for accurately and quantitatively assessing the IL-6/STAT3 pathway activation status in a biological sample or individual would be beneficial given the apparent role of this pathway in different disease conditions. Particularly, given its involvement in numerous biological functions and diseases, a gene expression signature-based readout of IL-6/STAT3 pathway activation may be more appropriate and predictive than relying on a single indicator of pathway activity, as the same signature of gene expression may be elicited by activation of multiple components of the pathway.